This invention relates to active derivatives of poly(ethylene glycol) and related hydrophilic polymers with a reactive moiety at one end of the polymer chain suitable for chemical coupling to another molecule.
Chemical attachment of the hydrophilic polymer poly(ethylene glycol)(PEG), which is also known as poly(ethylene oxide) (PEO), to molecules and surfaces is of great utility in biotechnology. In its most common form PEG is a linear polymer terminated at each end with hydroxyl groups:
HOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94OH 
This polymer can be represented in brief form as HOxe2x80x94PEGxe2x80x94OH where it is understood that the xe2x80x94PEGxe2x80x94 symbol represents the following structural unit:
xe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94
In typical form n ranges from approximately 10 to approximately 2000.
PEG is commonly used as methoxy-PEGxe2x80x94OH, or mPEG, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to ready chemical modification.
CH3Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94OH mPEG 
PEG is also commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, pentaerythritol and sorbitol. For example, the four-arm, branched PEG prepared from pentaerythritol is shown below:
C(CH2xe2x80x94OH)4+n C2H4Oxe2x86x92C[CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94OH]4 
The branched polyethylene glycols can be represented in general form as R(xe2x80x94PEGxe2x80x94OH)n in which R represents the central xe2x80x9ccorexe2x80x9d molecule, such as glycerol or pentaerythritol, and n represents the number of arms.
PEG is a well known polymer having the properties of solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity. One use of PEG is to covalently attach the polymer to insoluble molecules to make the resulting PEG-molecule xe2x80x9cconjugatexe2x80x9d soluble. For example, Greenwald, Pendri and Bolikal in J. Org. Chem., 60, 331-336 (1995) have shown that the water-insoluble drug taxol, when coupled to PEG, becomes water soluble.
In related work, Davis et al. in U.S. Pat. No. 4,179,337 have shown that proteins coupled to PEG have enhanced blood circulation lifetime because of reduced rate of kidney clearance and reduced immunogenicity. Hydrophobic proteins have been described that gain increased water solubility upon coupling to PEG. These applications and many leading references are described in the book by Harris (J. M. Harris, Ed., xe2x80x9cBiomedical and Biotechnical Applications of Polyethylene Glycol Chemistry,xe2x80x9d Plenum, New York, 1992).
To couple PEG to a molecule such as a protein or on a surface, it is necessary to use an xe2x80x9cactivated derivativexe2x80x9d of the PEG having a functional group at the terminus suitable for reacting with some group on the surface or on the protein (such as an amino group). Among the many useful activated derivatives of PEG is the succinimidyl xe2x80x9cactive esterxe2x80x9d of carboxymethylated PEG as disclosed by K. Iwasaki and Y. Iwashita in U.S. Pat. No. 4,670,417. This chemistry can be illustrated with the active ester reacting with amino groups of a protein (the succinimidyl group is represented as NHS and the protein is represented as PROxe2x80x94NH2):
PEGxe2x80x94Oxe2x80x94CH2xe2x80x94CO2xe2x80x94NHS+PROxe2x80x94NH2xe2x86x92PEGxe2x80x94Oxe2x80x94CH2xe2x80x94CO2xe2x80x94NHxe2x80x94PRO 
Problems have arisen in the art. Some of the functional groups that have been used to activate PEG can result in toxic or otherwise undesirable residues when used for in vivo drug delivery. Some of the linkages that have been devised to attach functional groups to PEG can result in an undesirable immune response. Some of the functional groups do not have appropriate selectivity for reacting with particular groups on proteins and can tend to deactivate the proteins.
Attachment of a PEG derivative to a substance can have a somewhat unpredictable impact on the substance. Proteins, small drugs, and the like can have less biological activity when cojugated with a PEG derivative. For others, activity is increased.
Another example of a problem that has arisen in the art is exemplified by the succinimidyl succinate xe2x80x9cactive esterxe2x80x9d mPEG-SS (the succinimidyl group is represented as NHS): 
The mPEG-SS active ester is a useful compound because it reacts rapidly with amino groups on proteins and other molecules to form an amide linkage (xe2x80x94COxe2x80x94NHxe2x80x94). A problem with the mPEG-SS active ester, which was recognized by K. Iwasaki and Y. Iwashita in U.S. Pat. No. 4,670,417, is that this compound possesses an ester linkage in the backbone that remains after coupling to an amine such as a protein (represented as PROxe2x80x94NH2):
mPEG-SS+PROxe2x80x94NH2xe2x86x92mPEGxe2x80x94O2Cxe2x80x94CH2CH2xe2x80x94CONHxe2x80x94PRO 
The remaining ester linkage is subject to rapid hydrolysis and detachment of PEG from the modified protein. Too rapid hydrolysis can preclude use of a PEG derivative for many applications. Several approaches have been adopted to solve the problem of hydrolytic instability. For example, mPEG succinimidyl carbonate has been proposed, which contains only ether linkages in the polymer backbone and reacts with proteins to form a conjugate that is not subject to hydrolysis.
It would be desirable to provide alternative PEG derivatives that are suitable for drug delivery systems, including delivery of proteins, enzymes, and small molecules, or for other biotechnical uses. It would also be desirable to provide alternative PEG derivatives that could enhance drug delivery systems or biotechnical products.
The invention provides chemically active polyethylene glycols and related polymers that are suitable for coupling to other molecules to give water-soluble conjugates, and in which the linkage between the polymer and the bound molecule is subject to predetermined cleavage for controlled delivery of the bound molecule into the surrounding environment.
The PEG and related polymer derivatives of the invention contain weak, hydrolytically unstable linkages near the reactive end of the polymer that provide for a sufficient circulation period for a drug-PEG conjugate and then hydrolytic breakdown of the conjugate and release of the bound molecule. Methods of preparing the active PEGs and related polymers, PEG conjugates, and methods of preparing the PEG conjugates are also included in the invention.
The PEG and related polymer derivatives of the invention are capable of imparting water solubility, size, slow rate of kidney clearance, and reduced immunogenicity to the conjugate, while also providing for controllable hydrolytic release of the bound molecule into the aqueous environment by design of the linkage. The invention can be used to enhance solubility and blood circulation lifetime of drugs in the blood stream and then to deliver a drug into the blood stream substantially free of PEG. In some cases, drugs that previously had reduced activity when permanently conjugated to PEG can have therapeutically suitable activity when coupled to a degradable PEG in accordance with the invention.
In general form, the derivatives of the invention can be described by the following equations: 
In the above equations,
xe2x80x9cPolyxe2x80x9d is a linear or branched polyethylene glycol of molecular weight from 300 to 100,000 daltons. Poly can also be a related nonpeptidic polymer as described in the Detailed Description;
n is the number of chemically active end groups on Poly and is the number of molecules that can be bound to Poly;
W is a hydrolytically unstable weak group;
T is a reactive group;
(Y-Pxe2x80x2)n represents a molecule for conjugation to Poly, in which Y is a reactive group that is reactive with T and Pxe2x80x2 is the portion of the molecule that is to be bound and released, including, for example, a peptide Pxe2x80x2 in which Y is an amine moiety and T is a PEG activating moiety reactive with amine moieties;
X is the new linkage formed by reaction of Y and T; and
G and I are new groups formed by hydrolysis. of W.
Examples of hydrolytically unstable groups W include carboxylate esters, phosphate esters, acetals, imines, orthoesters, peptides and oligonucleotides. T and Y are groups reactive toward each other. There are many examples of such groups known in organic chemistry. Some examples include active esters reacting toward amines, isocyanates reacting toward alcohols and amines, aldehydes reacting toward amines, epoxide reacting toward amines, and sulfonate esters reacting toward amines. Examples of Pxe2x80x2 include peptide, oligonucleotide and other pharmaceuticals. Examples of X include amide from reaction of active esters with amine, urethane from reaction of isocyanate with hydroxyl, and urea from reaction of amine with isocyanate. Examples of G and I are alcohol and acid from hydrolysis of carboxylate esters, aldehyde and alcohol from hydrolysis of acetals, aldehydes and amine from hydrolysis of imines, phosphate and alcohol from hydrolysis of phosphate esters, amine and acid from hydrolysis of peptide, and phosphate and alcohol from hydrolysis of oligonucleotides.
An example of the invention is shown in the following equation for conjugation of methoxy-PEGxe2x80x94OH (mPEG) with a peptide drug and for hydrolytic release of the peptide drug. The weak linkage W in the conjugate is an ester group. 
The released peptide contains no mPEG. The released peptide contains an additional short molecular fragment, which is sometimes called a xe2x80x9ctagxe2x80x9d and is the portion of the linkage opposite the PEG from the hydrolytically unstable linkage.
Thus, the invention provides hydrolytically unstable linkages in activated PEGs and related polymers that are suitable for controlled delivery of drugs from conjugation with the PEG to the surrounding environment. Several types of linkages, including ester linkages, are suitable for use in the invention. However, the ester linkages of the invention, in contrast to mPEG-SS and mPEG-SG, provide for variation and control of the rate of hydrolytic degradation.
The foregoing and other objects, advantages, and features of the invention, and the manner in which the same are accomplished, will be more readily apparent upon consideration of the following detailed description of the invention taken in conjuntion with the accompanying drawings, which illustrates an exemplary embodiment.